Process for (A) separating biological/ligands from dilute solutions and (B) conducting an immunochromatographic assay thereof employing superparamagnetic particles throughout

ABSTRACT

Superparamagnetic (“SPM”) subunits of 1-30 nm average mean diameter (e.g. ferro fluid) subparticles are treated with a magnetically noninterfering substance capable of coating and covering them (e.g, BSA) and they spontaneously form agglomerates of about 100 nm to about 450 nm or higher average mean diameter and are then used to form complexes with target biological ligands such as viruses, contained in large volumes of liquid. The complexes are subjected to the gradient intensity of a strong magnetic field, and excess liquid is removed, where upon an immunochromatographic assay is conducted to determine the identity and/or amount of target ligand present, in which operation SPM particles that bonded to the ligand function as tags for ligand detection.

[0001] This invention relates to using the same superparamagneticparticles, as more particularly described hereinafter, to concentratebiological substances believed to be sparsely present in large volumesof fluids and as labelling agents for detecting the quantity of the samebiological molecules present in a fluid sample.

BACKGROUND OR THE INVENTION

[0002] Heretofore it has become common to use metallic particles havingsuperparamagnetic properties to concentrate biological ligands resent insmall amounts in large volumes of aqueous fluids, including fluids ofmammalian origin such as urine. These metallic particles are often oflarge size (typically in the order of 1-5 μm or larger in average meandiameter) such that they cannot move, or do not move sufficientlyreadily, through the matrices used for either flow-through tests orlateral flow immunochromatographic (“ICT”) tests such as those commonlyused currently in many commercially available diagnostic tests foridentifying disease causative pathogens. In instance, where such testsare to follow the initial concentration step, removal of thesuperparamagnetic particles used for concentration is necessary,followed by adding a target specific conjugate labelled with achemiluminescent, fluorescent or radioactive or tag or a tag such ascolloidal latex particles, colloidal gold, or another colloidal metalwhich couples to the biological ligand and aids in the detectionthereof. The need to remove superparamagnetic particles used in ligandconcentration and then subject the concentrated ligand to anidentification or quantification assay often poses problems. Forexample, quantification of the small amount of biological ligandobtained by concentration is rendered inaccurate if even a tiny fragmentof concentrated ligand clings to the particles used for concentration;by the same token, incomplete removal of a small fragment of a magneticparticle may disrupt a qualitative identification of the concentratedsample by setting up an interaction with the labelling agent chosen foruse in the subsequent identification test. Even in cases wheresuperparamagnetic particles are employed to concentrate biologicalligands present in a large volumes of fluid and the nature of thesubsequent identification procedure renders separation of thesuperparamagnetic particles unnecessary, these particles have heretoforebeen viewed in the art as irrelevant to the subsequent identificationstep.

[0003] Large sized superparamagnetic particles have been preferred forligand concentration work, because their large size (in the order of 1to 5 μm or more) increases the mass of material bound to the targetligand and allows the gradient field of a fixed magnet to effectseparation with ease. Much smaller particles have been used in someinstances but often the low mass of magnetic material that they impartto their target, requires the introduction of magnetizable columns,filters or screens as an aid to separating the target molecules from thesample.

[0004] Particles heretofore used as tags for detecting a biologicalligand (regardless of whether it has been subjected to a firstconcentration step) are usually quite small. As already noted, this isespecially true where rapid “flow-through” or lateral flow matriceshaving narrow pores are employed as solid phase substrates. Particularlyin the lateral flow ICT format, particles used as detection markers mustbe small enough to migrate through the pores of the matrices and reachthe immobilized binding partner of the biological ligand being detected.

[0005] The present invention is based on the discovery that there is aclass of superparamagnetic particles which are small enough to functionas tags for detection of biological ligands in ICT test formats wheresolid porous matrices are employed and also have a sufficiently largemagnetic moment to function effectively as ligand concentrationadjuvants. The capability of using the same particles for concentrationand separation of a target ligand from a large volume of liquid and astags for a qualitative ligand identification test or a similar test thatnot only identifies but quantifies the amount of ligand enables asignificant increase in the sensitivity of the pre-assay concentrationstep. At the same time, the separation of the target ligand frominterfering or inhibitory substances that may be present in the originalsample is enhanced, the awkward need for removing a magnetic label isavoided and so is the equally awkward need for introducing a secondlabel.

BRIEF DESCRIPTION OF THE INVENTION

[0006] The present invention utilizes superparamagnetic subunits of 1-30nm in average mean diameter, such as ferrofluid subparticles, which aremixed with bovine serum albumin (“BSA”) or a similar biologically andmagnetically non-interfering substance capable of coating and coveringsuch particles, whereby they form BSA-coated ferrofluid particles whichspontaneously agglomerate to masses each containing a number offerrofluid subunit “cores” or nuggets, each completely surrounded byBSA. These BSA-ferrofluid agglomerates have been found to be highlyeffective, at overall particle average mean diameters of at least about100 nm ranging up to about 450 nm, and at times even higher, (1) asagents from concentrating and separating out target biological ligandsfrom large liquid volumes in which they are initially present in traceamounts and (2) as tags for enabling detection of these target ligands,for identity confirmation purposes and/or for quantification inICT-format assays.

BRIEF DESCRIPTION OF THE DRAWING

[0007]FIG. 1 hereof is a plot of magnetic signal measured in millivolts(mV) against Respiratory Syncytial Virus (“RSV”) lysate in milligramsper milliliter at sample volumes of 0.1 ml., 1.0 ml. and 10.0 ml.,respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0008] The vistas opened by the use of this invention are bestappreciated from a consideration of the fact that the commerciallyavailable ICT assays described in the copending, commonly assigned U.S.patent application Ser. Nos. 09/139,720 and 09/397,110, which are bothhighly sensitive and specific for the identifying presence of particulardisease-causing bacteria are successfully run with a few drops of testfluid—in the order of 100 microliters of urine, for example. Bacteriamolecules, however, are large in comparison to the molecules of, e.g.,viruses and various biochemical substances, the presence orconcentration of which my be indicative of a disease state or anotherabnormal condition in a human patient.

[0009] These smaller molecules often are widely dispersed in samples ofmammalian fluid, such as urine, with the result that the sample sizeadequate to enable detection of particular bacteria in the urine of aperson suffering from a disease of which those bacteria are causative,is too dilute to insure that smaller disease-causing molecules will beequally readily detectable.

[0010] By affording a means of concentrating the smaller molecules in aliquid sample prior to assaying for them, one is enabled to detect and,if desired, quantify, the presence in, e.g., human urine, of moleculesthat—if run in the assay format described in the aforementionedcopending applications, without preconcentration, could not be detectedwith high sensitivity and specificity and might not be detectable atall. Experience to date with pre-assay concentration usingsuperparamagnetic particles composed of ferrofluid 1-30 nm diametersubunits distributed in a BSA matrix, said composite particles havingaverage mean diameters of between at least about 100 nm and about 450 nmand coated with an antibody to the target molecule which attracts thetarget molecule and couples thereto, thereby effecting the desiredconcentration upon exposure to the gradient of a magnetic field, whenfollowed by an ICT assay for the target molecule which assay employs theaforesaid superparamagnetic particles as tags in the assay, hasdemonstrated a gain of approximately 2 logarithms of sensitivity to thetarget molecule over results heretofore attainable with methods whereinit was attempted to perform a conventional ICT assay for the targetmolecule on the original sample without concentration.

[0011] The superparamagnetic material used in the investigative workdescribed herein—i.e. ferrofluid core subunits of 1-30 nm diameterdispersed in a magnetically and biochemically inactive matrix of BSA—canbe substituted as to ferrofluid by any other metallic subunits of thissize range that exhibit superparamagnetic properties, including metalsand metallic oxides which exhibit spinel structure alone or incombinations with one another. As already noted other materials that aremagnetically inactive and in themselves biochemically unreactive withthe target ligand may readily be substituted for BSA.

[0012] The procedure for concentration of a target molecule in anaqueous medium (including a mammalian bodily fluid such as urine, blood,saliva, sputum, etc.,) renders it necessary that theBSA-superparamagnetic core agglomerates having a composite particlediameter of at least about 100 nm be first coated with a material whichis a binding partner for the target molecule. The coatedsuperparamagnetic particles are then immersed in the fluid and incubatedfor a period of at least 15 and often 30-40 or more, minutes. Complexesof superparamagnetic particles and target ligand are thereby formed.These complexes are sequestered from the bulk of liquid sample byexposure to the gradient of a magnetic field. The liquid is then removedby aspiration, decanting or any other convenient method the particlesare washed and dispersed in a volume of a suitable buffer that issmaller than the volume of the original sample. An ICT strip ofnitrocellulose or other bibulous material upon which a stripe of bindingpartner for the target molecule—which may be the same one used in theconcentration step or a different one, depending upon the functionalityof the target molecule—has been immovably bound to the capture zonearea, contained in a “dipstick” ICT device format, is immersed in thebuffered dispersion of superparamagnetic particles complexes. Uponmigration of these particles complexes along the strip, the targetmolecule on their outer surface binds to its binding partner in theimmovable stripe, causing superparamagnetic particles to accumulatealong the stripe. Experience has shown that immovable striping ofbinding partner for the target molecule multiple lines, spaced apartfrom one another along the end of the strip remote from the samplereceiving end, may be appropriate to ensure efficient capture of thetarget ligand in this assay. The magnetic signal of thesuperparamagnetic tag on the capture line or lines in millivolts, isread in a suitable instrument. The instrument used for the work shown inthe ensuing specific examples was a Magnetic Assay Reader IV unitobtained from Quantum Design, Inc., San Diego, Calif.

[0013] This unit is especially designed to be compatible with smallvolume assay formats, such as those which exhibit the end result as aline or lines of accumulated magnetic tag material. Because of thepermeability of the magnetic field of the superparamagnetic tag, signaldue to any analyte immobilized to the capture line is read as a singlemagnetic mass. This is in contrast to readings obtained from opticalinspection which detect only the surface appearance of the capture line.According to the manufacturer, the magnetic reading is linear withrespect to the mass of magnetic material on the capture line through atleast four orders of magnitude. The construction of standard curvescorrelating measured magnetic signal to target ligand amount is readilyachievable by methodology that is well known in the art.

[0014] It is anticipated that, for concentrating target moleculespresent in mammalian bodily fluids, such as, e.g. urine, saliva, blood,etc. at very high dilution levels, it may at times be necessary to makeuse of auxiliary magnetizable columns, filters or screens, or theaddition of nickel powder to the sample, to facilitate completeseparation from the sample and from unbound particles, of the low massof superparamagnetic material actually bound to target molecules.

[0015] The following examples, which are illustrative only and in nosense limiting, illustrate how the invention works in practice:

EXAMPLE 1

[0016] A partially purified viral lysate of respiratory syncytial virus(“RSV”) obtained from Chemicon (Catalog #Ag857, Lot 21031072) wasdiluted in an aqueous buffer of pH 7.8+0.1 having the followingcomposition:

[0017] Tris base—24.22 grams per liter (g.p.l.)

[0018] Triton X-100—10 ml./liter

[0019] T ween 20—10 ml./liter

[0020] N-tetradecyl-N, N-dimethyl-3-ammonio-1-propane sulfonate—20.0g.p.l.

[0021] Sodium azide—0.2 g.p.l.

[0022] Water added to make 1 liter

[0023] The resulting dilution contained 0.05 mg/ml. of RSV lysate.Samples of 0.1 ml., 1 ml. and 10 ml., respectively of this dilution werecarefully withdrawn after thorough mixing. To each of the 3 samplesthere was then added 5 microliters of approximately 250 nm average meandiameter superparamagnetic particles consisting of ferrofluid subunitsof 1-30 nm diameter embedded in and each separately surrounded by BSA,which composite particles had been previously coated with anti-RSVmonoclonal antibody obtained from Viro Stat, Inc. (Catalog #0631, LotRM286). The mixture of the coated superparamagnetic particles andbuffered viral lysate was in each instance thoroughly mixed and allowedto incubate for 30 minutes at room temperature on a blood bag rotatorplatform. Each sample was then exposed to the gradient magnetic fieldintensity produced by a strong rare earth permanent magnet and heldstationary for at least 30 minutes, thereby concentrating thesuperparamagnetic conjugate and any bound RSV lysate and sequesteringthem in the area of greatest field intensity proximal to the magnet. Ineach instance the supernatant was then removed by aspiration and 100microliters of the above-described buffer was then added.

[0024] Each of the three resulting sample concentrates was thoroughlymixed and placed in contact with a 22.5 mm wide nitrocellulose lateralflow ICT membrane (purchased from Millipore Corp. and identified asHF07504, Lot RK 000231) upon which had earlier been immovably stripedacross its width in the “capture” zone (located in the area most remotefrom the point of sample introduction) the same anti-RSV-monoclonalantibody referred to above. The contact with the nitrocellulosemembrane, in each instance was initiated through an absorbent bridgingpad, whereby the magnetic complex with analyte bound thereto was causedto migrate laterally through the strip to the capture line, along whichmagnetic conjugate bound to the viral lysate analyte bound, but magneticconjugate free of viral lysate did not bind and was allowed to flow intoan absorbent zone positioned after the capture line. In each instancethe magnetic signal in millivolts of the capture line was read with theQuantum Design instrument referred to above. The exact procedure wasrepeated for each sample using run buffer alone, without the present ofRSV viral lysate, as a negative control. The measured results are shownin the following Table 1: TABLE #1 Measured Signal in mV RSV LysateConcentration 0.1 ml 1.0 ml 10 ml. in mg 1 ml sample sample sample 0 7.317.6 12.60 0.05 41.2 218.2 408.20

[0025] The results set forth in Table 1 are graphed in FIG. 1 hereof,wherein the intensity of magnetically induced signal is represented as astraight line function of RSV lysate concentration in mg./ml. for eachsample volume assayed.

[0026] The signal to noise ratio and the detection limit in mg./ml. foreach sample were calculated and the results appear in the followingTable 2: TABLE #2 RSV Lysate Concentration 0.05 mg. per ml. DetectionLimit in Signal to Noise Ratio mg./ml. 0.1 ml. 5.6 0.027 sample 1.0 ml.12.5 0.012 sample 10 ml. 32.4 0.0046 sample

[0027] Later work has shown that the total sensitivity of antigenicdetection for each sample volume can be increased if multiple fixedstripes of antibody are applied to the ICT membrane, each spaced apartfrom one another along the sample flow path and the total magneticmoment of these capture lines is measured.

[0028] This example as presented illustrates the efficacy andfeasibility of the superparamagnetic particles of this invention, whencoated with an appropriate biological ligand such as the antibodyemployed in the work underlying this example, and thus enablingextraction of a biological ligand from a dilute solution in which itoccurs and thereby also concentrating the ligand, It also illustratesthe efficacy and practicality involved in using the samesuperparamagnetic particles as tags for the biological ligand in anensuing ICT assay.

EXAMPLE 2

[0029] This example involves a possible use of the superparamagneticparticles described herein in an experimental ICT test for quantifyingLegionella pneumophila serogroup 1 in environmental water. The presentcommercially available test is described in copending, commonly assignedU.S. application Ser. No. 09/458,998 filed Dec. 10, 1999 as acontinuation-in-part of copending, commonly assigned U.S. applicationSer. No. 09/139,770 filed Aug. 25, 1998.

[0030] In this experiment, two 100 ml. samples of cooling tower waterwere drawn at Hood Dairy, Portland, Me. and held at temperature of 2-8°C. To one of these samples was added sufficient Legionella pneumophilaserogroup 1 bacteria to enrich the sample bacteria content by 95colony-forming units (“CFU”) per ml. Both samples were subjected to afiltration concentration on a small pore membrane as described in detailin copending U.S. application Ser. No. 09/458,998. The particulateretained on the membrane was recovered on a swab in each instance andwas reconstituted to a sample volume of 200 μl with a buffer compositioncomposed of aqueous 0.05 M Tris HCl and 2.5% Tween 20 having a pH of7.0±0.1. Each sample was then carefully split into two portions. Allfour resulting samples were of equal volume.

[0031] Particles of approximately 100 nm average mean diameter composedof ferrofluid subunits, each of 1-30 nm average mean diameter,distributed in an enveloping matrix of BSA were coated withanti-Legionella pneumophila serogroup 1 antibodies which had beenpurified as described in copending U.S. application Ser. No. 09/139,720.Equal amounts of the resulting conjugate were added to all four of thesamples and allowed to incubate for 15 minutes. One sample having noadded Legionella pneumophila serogroup 1 bacteria and one samples having95 CFu/ml of added bacteria were immediately subjected to an ICT testperformed with a dipstick style lateral flow device comprising anitrocellulose membrane pretreated by the application of a fixed stripeof purified anti-Legionella pneumophila serogroup 1 antibodies at theend of the strip most remote from the point of sample-introduction. Thetest strips and sample in each instance were not washed; once thesamples had migrated to the end of the nitrocellulose membrane, theconductivities in millivolts (mV) of the capture lines were read by theQuantum Design instrument referred to above. The sample having no addedbacteria gave a negative reading of −248.4 mV, which is believedattributable to the presence in the sample of particulate that was notbroken down. The sample with added bacteria (95 CFU/ml) gave a readingof 375 mV.

[0032] The remaining two samples were each washed 3 times with 500 ml.of the buffer and then reconstituted to 100 μl and run in the samemanner in identical ICT test, to first two samples. The magnetic momentsof the capture lines of each of the respective test strips were read inthe Quantum Design instrument. The washed sample having no addedbacteria gave a reading of 8.5 mV. The washed sample with added bacteriagave a reading of 161.8 mV.

[0033] The example supports the broad concept of superparamagnetic allyseparating the target ligand—in this instance the O-polysaccharideantigen of Legionella pneumophila serogroup 1—from a liquid sample,using superparamagnetic particles, followed by conducting an immunoassayusing the same supermagnetic label for detection.

[0034] Those skilled in the art will recognize many opportunities formaking use of the particles and methods referred to herein beyond thepossibilities explicitly disclosed. It is therefore intended that thescope of this invention be limited only by the appended claims.

We claim: 1 Particles having an average mean diameter of at least about100 nm, composed of discrete subunits of from 1 to 30 nm in average meandiameter of superparamagnetic material which are separately spaced apartfrom one another within a covering matrix of non-magnetic, non-metallicmaterial that is compatible with, but in itself non-reactive with,target biological ligands, which particles, when coated with a bindingpartner to a target biological ligand contained in an aqueous fluid andexposed to such fluid, form complexes with the target biological ligand,which complexes, when exposed to the gradient of a magnetic field,become sequestered from the bulk of aqueous fluid and, when thereafterintroduced to the sample receiving end of a dipstick configuredimmunochromatographic (“ICT”) device comprising a strip of bibulousmaterial having at least one immovable stripe of a binding partner forthe biological ligand permanently affixed thereto at the end remote fromits sample receiving end, become permanently affixed to said immovablestripe when the target ligand reacts with its immovable binding partner,whereby the stripe exhibits a measurable magnetic moment, the intensityof which is correlatable, in a known manner, to the amount of saidtarget ligand recovered from said aqueous fluid. 2 Particles accordingto claim 1 wherein the discrete subunits of superparamagnetic magneticmaterial of from 1 to 30 nm in average mean diameter are discreteparticles of ferrofluid and the matrix of nonmagnetic, non-metallicmaterial is bovine serum albumen. 3 A process wherein particles as setforth in claim 1 (a) are coated with a target biological binding partnerfor a biological ligand known to be, or suspected of being, present inan aqueous fluid, (b) as so coated are then immersed in said fluid andincubated therewith for a time sufficient to enable any targetbiological ligand present to form complexes with said particles byreacting with its binding partner coated on said particles and (c) saidcomplexes are thereupon exposed to the gradient of a magnetic fieldwhereby the complexes are sequestered from the bulk of the aqueousfluid. 4 A process according to claim 3 wherein following thesequestration of the complexes formed in step (c), the aqueous fluid isremoved, the complexes are washed and dispersed in an aqueous buffer,and (d) the resulting dispersion is applied to the sample receiving endof a dipstick-configured ICT device comprising a strip of bibulousmaterial having at least one immovable stripe of a binding partner forsaid biological ligand permanently affixed thereto at the end remotefrom its sample receiving end, (e) the dispersion migrates along saidstrip to said immovable stripe where the complexes, which have saidtarget biological ligand bound thereto permanently bind to said stripe;(f) the magnetic intensity of the stripe is measured and (g) thatintensity is correlated in a known manner to the amount of saidbiological ligand removed from the liquid to which the particles wereoriginally exposed 5 A process according to claim 4 wherein multipleimmovable stripes of the same binding partner for said biological ligandhave been permanently affixed at the end that is remote from the samplereceiving end of the strip of bibulous material and they are in spacedapart relationship from one another, whereby (a) as the complexes flowalong the strip, some portion of the target biological ligand thereinbinds to each of said stripes (b) the magnetic moment of each stripe ismeasured, (c) the respective magnetic moments of the stripes aretotalled and (d) the total magnetic moment is correlated in a knownmanner to the amount of target ligand originally present in the aqueousliquid to which the said particles were first exposed. 6 A processaccording to claim 4 in which the aqueous fluid is an aqueousenvironmental sample. 7 A process according to claim 4 in which theaqueous fluid is of mammalian origin. 8 A process according to claim 7in which the aqueous fluid is urine. 9 A process according to claim 4wherein the biological binding partner to the target ligand initiallycoated on the particles is the same as the biological binding partnerthat is immovably striped on the strip of bibulous material. 10 Aprocess according to claim 4 wherein the biological binding partner tothe target ligand initially coated on the particles is different fromthe biological binding partner to that ligand which is immovably stripedon of the strip of bibulous material. 11 A process wherein particles asset forth in claim 2 are: (a) are coated with a target biologicalbinding partner for a biological ligand known to be, or suspected ofbeing, present in an aqueous fluid, (b) as so coated are then immersedin said fluid and incubated therewith for a time sufficient to enableany target biological ligand present to form complexes with saidparticles by reacting with its binding partner coated on said particlesand (c) said complexes are thereupon exposed to the gradient of amagnetic field whereby the complexes are sequestered from the bulk ofthe aqueous fluid. 12 A process according to claim 11 wherein followingthe sequestration of the complexes formed in step (c), the aqueous fluidis removed, the complexes are washed and dispersed in an aqueous buffer,and (d) the resulting dispersion is applied to the sample receiving endof a dipstick-configured ICT device comprising a strip of bibulousmaterial having at least one immovable stripe of a binding partner forsaid biological ligand permanently affixed thereto at the end remotefrom its sample receiving end, (e) the dispersion migrates along saidstrip to said immovable stripe where the complexes, which have saidtarget biological ligand bound thereto permanently bind to said stripe;(f) the magnetic intensity of the stripe is measured and (g) thatintensity is correlated in a known manner to the amount of saidbiological ligand removed from the liquid to which the particles wereoriginally exposed. 13 A process according to claim 12 wherein multipleimmovable stripes of the same binding partner for said biological ligandhave been permanently affixed at the end that is remote from the samplereceiving end of the strip of bibulous material and they are in spacedapart relationship from one another, (a) whereby as the complexes flowalong the strip, some portion of the target biological ligand thereinbinds to each of said stripes, (b) the magnetic moment of each stripe ismeasured, (c) the respective magnetic moments of the stripes aretotalled and (d) the total magnetic moment is correlated in a knownmanner to the amount of target ligand originally present in the aqueousliquid to which the said particles were first exposed. 14 A processaccording to claim 11 in which the aqueous fluid is an aqueousenvironmental sample. 15 A process according to claim 11 in which theaqueous fluid is of mammalian origin. 16 A process according to claim 15in which the aqueous fluid is urine. 17 A process according to claim 11wherein the biological binding partner to the target ligand initiallycoated on the particles is the same as the biological binding partnerthat is striped on the strip of bibulous material. 18 A processaccording to claim 11 wherein the biological binding partner to thetarget ligand initially coated on the particles is different from thebiological binding partner to that ligand which is immovably striped onof the strip of bibulous material. 19 Particles according to claim 1whose the average mean diameter is between about 100 nm and about 450nm. 20 Particles according to claim 2 whose average mean diameter isbetween about 100 nm and about 450 nm.